Selection of a multidrug resistance plasmid by sublethal levels of antibiotics and heavy metals

Abstract

How sublethal levels of antibiotics and heavy metals select for clinically important multidrug resistance plasmids is largely unknown. Carriage of plasmids generally confers substantial fitness costs, implying that for the plasmid-carrying bacteria to be maintained in the population, the plasmid cost needs to be balanced by a selective pressure conferred by, for example, antibiotics or heavy metals. We studied the effects of low levels of antibiotics and heavy metals on the selective maintenance of a 220-kbp extended-spectrum β-lactamase (ESBL) plasmid identified in a hospital outbreak of Klebsiella pneumoniae and Escherichia coli. The concentrations of antibiotics and heavy metals required to maintain plasmid-carrying bacteria, the minimal selective concentrations (MSCs), were in all cases below (almost up to 140-fold) the MIC of the plasmid-free susceptible bacteria. This finding indicates that the very low antibiotic and heavy metal levels found in polluted environments and in treated humans and animals might be sufficiently high to maintain multiresistance plasmids. When resistance genes were moved from the plasmid to the chromosome, the MSC decreased, showing that MSC for a specific resistance conditionally depends on genetic context. This finding suggests that a cost-free resistance could be maintained in a population by an infinitesimally low concentration of antibiotic. By studying the effect of combinations of several compounds, it was observed that for certain combinations of drugs each new compound added lowered the minimal selective concentration of the others. This combination effect could be a significant factor in the selection of multidrug resistance plasmids/bacterial clones in complex multidrug environments. Importance: Antibiotic resistance is in many pathogenic bacteria caused by genes that are carried on large conjugative plasmids. These plasmids typically contain multiple antibiotic resistance genes as well as genes that confer resistance to biocides and heavy metals. In this report, we show that very low concentrations of single antibiotics and heavy metals or combinations of compounds can select for a large plasmid that carries resistance to aminoglycosides, β-lactams, tetracycline, macrolides, trimethoprim, sulfonamide, silver, copper, and arsenic. Our findings suggest that the low levels of antibiotics and heavy metals present in polluted external environments and in treated animals and humans could allow for selection and enrichment of bacteria with multiresistance plasmids and thereby contribute to the emergence, maintenance, and transmission of antibiotic-resistant disease-causing bacteria.

FIG 1

Selection coefficients as function of antibiotic concentrations during competition experiments between strains carrying the pUUH239.2 plasmid and susceptible strains at low levels of antibiotics and heavy metals. Competitions in the presence of tetracycline (A), trimethoprim (B), erythromycin (C), kanamycin (D), arsenite (E), and copper (F). Data can be found in Table S3 in the supplemental material. Standard deviations are indicated.

FIG 2

Selection coefficients as a function of antibiotic and heavy metal concentrations during competition experiments between resistant strains carrying the pUUH239.2 plasmid and susceptible plasmid-free strains. (A) Competitions in the presence of combinations of trimethoprim and erythromycin. The total normalized concentration is the sum of the two normalized concentrations, where the concentration of each compound was first normalized to its respective MSC. Data can be found in Table S4 in the supplemental material. (B) Competitions in the presence of combinations of arsenic and tetracycline. These competitions were performed in LB medium, explaining why the selection coefficients of tetracycline in this experiment were different from the values in Fig. 1A, where Mueller-Hinton medium was used. Data can be found in Table S5. The differences in MSC with and without arsenic were significant (P value = 0.0001 in a paired t test). (C) Competitions in the presence of combinations of arsenic, tetracycline, and trimethoprim. The total normalized concentration is the sum of all normalized concentrations, where the concentration of each compound was first normalized to its respective MSC. Data can be found in Table S6. Standard deviations are indicated.

FIG 3

Selection coefficients as function of antibiotic concentrations during competition experiments between strains carrying chromosomally carried resistance genes and susceptible strains at low levels of antibiotics. Competitions in the presence of tetracycline (A), trimethoprim (B), and erythromycin (C). Data can be found in Table S7 in the supplemental material. Standard deviations are indicated.